Aging and Endothelial Cell Function

描述（申请人提供）：血管生成随着年龄的增长而受损。这损害了伤口的修复和缺血器官的血运重建。微血管内皮细胞（mECs）的迁移受到抑制是年龄相关的血管生成障碍的核心组成部分之一。在三维（3D）胶原中，mec的运动受与细胞表面相关的基质金属蛋白酶（MMP2、MT1-MMP）及其主要抑制剂TIMP2的调控。此外，老年人类mec的迁移和小管形成减少与TIMP2增加和MMP2/MT1-MMP活性降低有关。目前，在老年mECs中介导TIMP2过量和MMP2/MT1-MMP活性不足的机制尚不清楚；然而，初步数据表明一氧化氮（NO）是一个候选的调节因子。NO，一种血管活性的，TIMPs和MMPs的“上游”调节剂，在衰老的细胞和组织中减少。我们的假设是，一氧化氮水平的降低是TIMP2过剩和MMP活性不足的原因，这反过来又抑制了老年mec的迁移和小管形成。因此，老年mec中NO水平升高会导致MMP活性相应升高，从而促进迁移和小管发生。目标1和2

DESCRIPTION (provided by applicant): Angiogenesis is impaired in aging. This compromises the repair of wounds and revascularization of ischemic organs. One of the central components of age-associated impairment of angiogenesis is inhibited migration of microvascular endothelial cells (mECs). In 3-dimensional (3D) collagen, movement of mECs is regulated by matrix metalloproteinases (MMPs) associated with the cell surface (MMP2, MT1-MMP) and their primary inhibitor, TIMP2. Moreover, diminished migration and tubulogenesis by aged human mECs is associated with increased TIMP2 and decreased MMP2/MT1-MMP activity. Currently, the mechanism that mediates excess TIMP2 and deficient MMP2/MT1-MMP activity in aged human mECs is not understood; however, preliminary data implicate nitric oxide (NO) as a candidate regulatory factor. NO, a vasoactive, "upstream" modulator of TIMPs and MMPs, is decreased in aged cells and tissues. Our hypothesis is that diminished levels of NO are responsible for excess TIMP2 and deficient MMP activity that, in turn, inhibits the migration and tubulogenesis of aged mECs. Consequently, increasing NO levels in aged mECs will lead to corresponding increases in MMP activity, thereby improving migration and tubulogenesis. AIMs 1 and 2 will utilize human mECs (hmECs) from 7 young (mean age=26+6yrs) and 8 aged (mean age=67+11 yrs) donors; all cells are cultured in 3D collagen gels- a simulator of interstitial ECM in vivo. AIM 1 will define changes in synthesis and activity of MMP2/MT1-MMP/TIMP2 by hmECs in response to NO. AIM 2 will examine the functional consequences of exogenous and paracrine (from iNOS transduced fibroblasts) N)-induced changes on the ability of hmECs to migrate and undergo tubulogenesis. AIMs 3 and 4 will define NO effects on EC function and MMP2/MT1-MMP/TIMP2 activity during angiogenesis ex vivo and in vivo in young and aged F1 hybrid mice using two complementary models: AIM 3: formation of sprouts from explanted microvessels cultured ex vivo in 3D collagen, and AIMs 3b and 4: vascular ingrowth into polyvinyl alcohol (PVA) sponges implanted subcutaneously in vivo. The models of murine angiogenesis permit vascular growth to be studied and manipulated in animals that are matched in all respects except for age. In summary, this proposal will use NO to define mechanism(s) of MMP2/MT1-MMP/TIMP2 regulation and subsequent impairment of endothelial cell function in aging. The elucidation of mechanisms that regulate MMP activity in aged cells and tissues will assist the development of therapies to improve wound repair and revascularization of ischemic organs.